Sequencing of the human genome in a timely and cost effective manner will require improved instrumentation. The goal of this project is to enhance the performance of existing Marshfield SCAnning FlUorescence Detectors (SCAFUDs) for the purpose of DNA sequencing. SCAFUDs are used to detect fluorescently labeled DNA fragments as they migrate through 144 lane, vertical, polyacrylamide denaturing slab gels. Fluorescent signals from the detectors are arrayed into computer image files which are then manipulated using a suite of image analysis programs. Existing SCAFUDs have a maximum theoretical output for sequencing of 63 x 10-6 nucleotides per year. Improvements described in this proposal will boost the maximum theoretical output to at least 215 x 10-6 nucleotides per year. The approximately 3.5 fold increase in sequencing performance will be achieved through a number of incremental hardware and software improvements. Instrument design will be consolidated and modularized to reduce failure rate and to simplify repair. A new less massive and more light tight cover for the instrument will be developed. Autofocusing will be implemented to reduce instrument variability, operator error, and to improve laser safety. Adjustable gain amplifiers will be designed and attached to each detector. The excitation laser light will be introduced into the gel through Brewster's angle as compared to the current epi- illumination design. Polarization and filtering of the input laser light will be tested as a means of reducing background light reaching the detectors. The amount of sequence information obtained from each SCAFUD run will be increased by collecting data during both the forward and return passes of the laser head. Gel thickness, length, composition, and temperature will be varied to increase the rate at which quality sequence information can be collected. The numbers of dyes which can be detected simultaneously will be increased by addition of a second and, possibly, third laser source and by significantly simplifying the detection system. Extensive use will be made of new multichannel detector arrays. Image analysis software will be modified as necessary to pace the hardware changes. Minimization of sequencing cost will be the overriding factor used in evaluation of all instrument modifications. Existing and future image analysis and base calling software will be generalized and documented to facilitate transfer of this software to other research laboratories. Transfer of the SCAFUD hardware technology to other research laboratories will be accomplished either through marketing of a similar instrument by a private firm and/or through release of detailed construction drawings and parts lists for the instrument from Marshfield.